This invention relates to the field of in vitro radioimmunoassays and, more particularly, to an improved radioimmunological method for assaying plasma renin activity.
Hypertensive disease is often associated with high levels of aldosterone in the blood. In primary aldosteronism, this condition results from disease directly involving the adrenal gland. In renal vascular disease, on the other hand, high aldosterone levels may result from high concentrations of angiotensin II in in the blood. Angiotensin II is a potent vasopressor and stimulates production of aldosterone by the adrenal gland. Thus, in both primary aldosteronism and renal vascular disease, high levels of aldosterone may be observed in the blood and aldosterone level, as such, does not provide a basis for distinguishing between these two different causes of hypertension.
It is well recognized that primary aldosteronism can be distinguished from angiotensin II-induced aldosterone secretion by measurement of plasma renin activity. Renin is a proteolytic enzyme secreted by juxtaglomerular cells in the kidney. Angiotensinogen, an .alpha.-2-globulin produced by the liver, is converted by renin to the decapeptide angiotensin I. Although itself biologically inactive, angiotensin I is converted to angiotensin II in the pulmonary circulation. In patients suffering from renal hypertension, the plasma renin activity, and thus the angiotensin generation rate, is high, while in primary aldosteronism renin activity is low.
Bioassay techniques are known for the determination of plasma renin activity. The convenience of making renin acitivity determinations has been greatly enhanced, however, by the development of radioimmunoassay techniques for its measurement. Radioimmunoassay is a method based on the phenomenon of competitive protein binding in which a reaction system is prepared containing a known amount of radioactively labeled antigen, an unknown amount of unlabeled antigen to be assayed, and a standard amount of an antibody for the antigen. The system is incubated at a predetermined temperature for a predetermined time sufficient for the competitive reaction of the labeled and unlabeled antigen with the antibody to proceed to an analytically significant extent. The proportion of labeled antigen bound to antibody in the assay reaction system is then determined by radioactive counting. This determination also establishes the proportion of unlabeled antigen bound to antibody and, by inference, the amount of unlabeled antigen initially charged to the reaction system.
Competitive protein binding assays are conventionally illustrated by the following reaction equations:
Ag* + Ab.revreaction. Ag*Ab + Ag AgAb
where
Ag* = radioactively labeled antigen PA0 Ag = unlabeled antigen PA0 Ab = antibody specific for Ag/Ag* PA0 Ag*Ab = labeled antigen/antibody complex PA0 AgAb = unlabeled antigen/antibody complex
Provided that the total of labeled and unlabeled antigens is in excess, unlabeled antigen from an unknown sample competes with the labeled antigen for the binding sites on the antibody. For given quantities of antibody and labeled antigen, the proportion of labeled antigen bound to the antibody decreases with increasing amounts of unlabeled antigen in the reaction system. Using standard known quantities of unlabeled antigen, data may be developed correlating the ratio of bound to free labeled antigen with the amount of unlabeled antigen in the reaction system. A curve developed from such data may then be used to determine the quantities of the unlabeled antigen in a test reaction system to which a sample containing an unknown quantity of unlabeled antigen has been charged.
Since no radioimmunoassay methods are presently available for directly determining the quantity of renin in a plasma sample, renin activity is measured in terms of the rate of angiotensin I generation in the sample. Methods are available in which a plasma sample is divided, one portion kept in the cold to prevent significant angiotensin I generation and the other sample incubated at elevated temperature, for example, body temperature, to cause generation of angiotensin I by action of renin on angiotensinogen. Inhibitors are included in the generation mixture to prevent either formative of angiotensin II by action of converting enzymes on angiotensin I or destruction of angiotensin I by various proteolytic enzymes. After the generation step is complete, both the generated sample and the base sample which had been kept in the cold are subjected to radioimmunoassay for determination of angiotensin I content. The difference between the angiotensin I content of the generated sample and that of the base sample may be divided by the time of generation in order to determine renin activity in terms of angiotensin I generated per unit volume per unit time.
A serious drawback of previously available radioimmunoassay methods for measurement of renin activity is the necessity of carrying out the radioimmunoassay step in the cold, typically at 4.degree.C. Because of the presence of renin in both the base sample and the generated sample, it has been thought necessary to use such low temperatures in order to avoid error introduced by generation or destruction of angiotensin I during the radioimmunoassay. As a consequence of the low temperatures which are used, the time required for adequate reaction is long, for example, 18-24 hours. Such lengthy incubation times are obviously inconvenient and render the assay more expensive.
An unfulfilled need has, therefore, existed for an improved radioimmunoassay method for determining plasma renin activity which does not require the extensive incubation period heretofore considered necessary to avoid incidental angiotensin I generation.